A major goal of developmental research has been the understanding of the mechanisms by which cells become committed to particular fates and the sequence of events through which they then differentiate into morphologically and functionally distinct cell types. In the mammalian embryo, the visceral yolk sac (VYS) is the first morphologic and functional development of two interrelated tissues arising from newly proliferating mesoderm: blood cells and endothelial cells. Blood cells are functionally evident in the mouse embryo by day 6-7, and morphologically evident by day 8. Yet, nothing is known about the signals which trigger these events. The overall aim of this study is to begin to elucidate the molecular events underlying the initiation of hematopoiesis in the mammalian embryo. We hypothesize that genes transiently expressed during early tissue development may be involved with the process of lineage specification, rather than being the products of the differentiated phenotype. We will, therefore, isolate genes expressed during the initial stages of embryonic hematopoiesis by subtractive screening of a PCR amplified cDNA library. The spatial and temporal pattern of expression of these genes will be investigated by in situ hybridization and Northern blots. To identify the function of selected genes, full length transcripts will be sequenced and analyzed for functional motifs. Their expression will be investigated in both murine erythroleukemia cells (MELC) and the murine genetic mutant W, with altered hematopoiesis. Antisense oligomers of selected genes will be added to MELC and to embryonic stem cell cultures. If induced blood cell formation is blocked or altered, then a direct role for these genes in hematopoietic development can be postulated. The size of the protein products of these genes as well as their temporal and spatial patterns of expression will be determined by Western blots and immunostaining using antibodies raised to fusion proteins. This information will provide clues to their regulation as well as their function, eg. nuclear vs secreted proteins. We expect that these clones will be the earliest lineage-specific hematopoietic genes available. Even if they are not directly involved with lineage specification, they will be invaluable markers for the study of lineage development in the mouse, since no early markers for mesoderm or hematopoietic tissue as yet exist. The study of these genes may also provide a better understanding of clonal disorders such as CML and erythroleukemia.